Objective To investigate the influence of GraS（T136I） mutation on vancomycin resistance in vancomycin-intermediate Staphylococcus aureus（VISA） and its mechanism. Methods The homologous recombination technique was used to introduce the GraS（T136I） mutation site of XN108（VISA） into Newman（VSSA） to construct the mutant strain of Newman-GraS（T136I），and the same method was used to reverse the mutation in XN108 to construct the reverse strain XN108-GraS（I136T）. The E-test stripes were used to measure the resistance of each strain to vancomycin； a transmission electron microscope was used to observe the cell wall thickness of each strain；RT-qPCR was used to measure the changes in the expression levels of peptidoglycan synthesis-related genes on the cell wall of Staphylococcus aureus in XN108 and its reverse mutant strain XN108-GraS（I136T）；the LacZ report system was used to measure murC gene promoter activity；binding site analysis and electrophoretic mobility shift assay（EMSA） were used to confirm the regulatory effect of GraSR on the murC gene. Results The allelic replacement strains of Newman-GraS（T136I） and XN108-GraS（I136T） were constructed successfully. The minimal inhibitory concentration（MIC） of the reverse strain XN108-GraS（I136T） to vancomycin decreased from 12 μg/mL to 8 μg/mL，and the MIC of the mutant strain Newman-GraS（T136I） to vancomycin increased from 1.5 μg/mL in wild strain to 4 μg/mL. The transmission electron microscope showed that XN108-GraS（I136T） had a significantly thinner cell wall than the wild strain XN108［（20.097±2.862） nm vs.（40.283±3.784） nm，P=0.000］. RT-qPCR showed that the peptidoglycan synthesis-related genes murC，murD，and mraY on the cell wall of Staphylococcus aureus were significantly downregulated in the reverse strain XN108-GraS（I136T）（P=0.000），with the greatest reduction in murC（about 1.935 times）. The LacZ activity test showed that the promoter activity of the murC gene in XN108 was significantly higher than that in XN108-GraS（I136T）（2 182.333±104.580 vs. 1 593.333±179.258，P=0.008），and the binding site analysis and EMSA confirmed that GraR activated by GraS could directly bind to the control region of the murC gene. Conclusion GraS（T136I） mutation in Staphylococcus aureus can promote the formation of VISA，possibly by upregulating the expression of the key murC gene in cell wall synthesis and leading to cell wall thickening.